Project 2 Summary/Abstract Evaluating the dynamic protein-protein interactions mediated by LCDs within EWS/FLI and its partners in living cells via single molecule imaging (Program Leader: Darzacq; Collaborator: Tjian) Our goal is to characterize how low-complexity domain (LCD)-LCD specificity drives the formation of nuclear domains with distinct functional properties. In addition we will characterize the relationship between the emerging behavior of protein hubs and the function of single proteins at their cognate DNA targets. Understanding LCD-LCD interactions and their relation to nuclear organization and function will be an important first step toward learning how to intervene at these protein-protein interfaces as potential therapeutic targets. Our proposal exploits the cutting edge imaging technologies to study EWS/FLI function in the dynamic and complex environment of native chromatin in a living cell. This powerful multi-pronged strategy will reveal how the molecular functions of a key oncogene product (EWS/FLI) regulates gene expression and triggers neoplasia. The Darzacq lab will characterize the specificity of LCD-LCD interactions using their previously developed single locus system. An important part of the work will be to develop robust assays to identify locus specific enrichments of transcription factors using a candidate approach and then to characterize the effect of specific drugs on EWS/FLI hub composition and function. By creating the tools to characterize and measure LCD-LCD interactions we will empower the field with tools to design and screen effectors molecules emerging from the different screens performed by other members of the consortium. This potentially new platform for developing drug screens may require a rethinking of the assays and chemical libraries currently used in conventional drug screens.